3 3 4 dichloro phenyl 1 1 dimethylurea dcmu

Intact chloroplasts from 10 g of 12-day-old PsbO1^1–85GFP seedlings were isolated and resuspended in HMS buffer containing 10 μM chymostatin (BioShop) to inhibit serine proteases. Chloroplasts (0.2 mg ml^–1 chlorophyll) were incubated at 25 °C under 120 μmol m⁻² s⁻¹ white light or in the dark (wrapped by aluminum foil) for 12–60 min. Transport in the dark was induced by adding 5 mM Mg-ATP (BioShop) to intact chloroplasts. Reactions were stopped using HMS buffer containing EDTA to a final concentration of 10 mM. Alternatively, transport assays were conducted in the presence of 10 mM sodium azide, 10 μM geldanamycin (Sigma, G3381), 10 μM 3-(3,4-dichloro-phenyl)-1,1-dimethylurea (DCMU; Sigma-Aldrich), or 12 μM 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone (DBMIB; Sigma-Aldrich). DCMU and DBMIB were prepared as 1000-fold concentrated stocks in 95% ethanol prior to dilution in H₂O just prior to use.

The polyclonal anti-phosphothreonine (anti-pThr) antibodies were from Cell Signaling Technology™, Inc. (Danvers, MA; cat. 9381S) and Abcam (Cambridge, UK; cat. Ab9337). For loading controls and normalization in quantitation analyses either, a monoclonal anti-actin antibody (Cat. No. N350, originally purchased from Amersham) known to cross-react with actin from many species [21 (link)] including Symbiodiniaceae [20 (link), 22 (link)], or the purified IgG fraction (~ 1 mg/ml IgG as stock) of a rabbit anti-SBiP1 polyclonal antibody [17 (link)], were used. Alkaline-phosphatase (AP) conjugated polyclonal anti-rabbit IgG and anti-mouse IgG antibodies raised in goat were from Zymed^®-Life Technologies (Grand Island, NY). Reagents 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) were from Promega (Madison, WI). The inhibitor of photosystem II (PSII) 3-(3,4- dichlorophenyl)-1,1-dimethylurea (DCMU) was from Sigma. All other reagents were from Sigma.

For experiments with norflurazon (Sigma‐Aldrich), growth medium was supplemented with 5 μM norflurazon and 1% (w/v) sucrose to allow growth in the absence of photosynthesis (e.g. Fig. S3a). For bioluminescence imaging experiments with 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea (DCMU, Sigma‐Aldrich), 20 μM DCMU was added to the 100 μl of 5 mM luciferin that was dosed onto seedlings. For RNA sampling, 100 μl of 20 μM DCMU was dosed onto seedlings. In both cases, DCMU was dosed onto seedlings 24 h before the start of light treatment. Inhibitors were dissolved in dimethylsulfoxide (DMSO) (working concentrations of DMSO were 0.0025% (v/v) and 0.01% (v/v) with norflurazon and DCMU, respectively), and inhibitor controls contained an equal volume of DMSO without the inhibitor.